In previous work in collaboration with Dr. Minoru Ko (LG-NIA),we were able to establish a stem-cell differentiation assay based on cell morphology alone, using phase-contrast imaging without specific markers. This year we were able to extend this work in a collaboration with Noriko Saitoh of Kumamoto University in Japan to characterize the quality of human induced pluripotent stem (iPS) cells. As part of the same collaboration, we also published a study where we used pattern recognition in fluorescence microscopy of cultured human cels to show that the actin-related protein ARP6 affects the structure of the nucleolus. We have continued work characterizing the molecular basis of morphological age-state transitions during the C. elegans life-span. In published work we used WND-CHARM to identify distinct morphological aging states in C. elegans. We used this technique to sort worms based on their age state during a transition period where an aging population is evenly divided between individuals in Stage I and Stage II. The worms were identical genetically, by chronological age, by growth conditions, and by visual appearance, and could only be sorted into age-states using WND-CHARM. Micro-array experiments performed on these two sub-populations revealed several hundred genes with significantly altered expression profiles. By comparing our gene lists with those from other aging studies in C. elegans, we were able to identify several gene families and functional groups that were unique to our study. A prevailing theme of the aging genes uniquely identified in this study were those involved in targeted proteolysis, which appears to be a hallmark of this first aging state transition. In previous work, we have developed an age-state classifier that enables us to more robustly identify the aging state occupied by individual worms. More recently we have shown that this state-classifier is robust enough to identify these states in other strains of worms. We used this classifier to manually sort several thousand worms by age-state in order to study gene expression patterns specific to these aging states. Currently, we have completed work on expression profiling of all three observed aging states and transitions in C. elegans. Preliminary results indicate that the biological replicates we performed in quadruplicate were reproducible, and that there are many reproducible differences in gene expression in the transitions between age-states. We are currently analyzing these gene expression results. We have begun to characterize the effect of RNAi knockdown on the genes identified as differentially expressed in the first age-state transition. A group of genes producing secreted proteins with cysteine-rich regions appears to contain genes that can either advance or delay the first transition. We are also looking at a set of heat-shock genes who's expression is among the most significantly altered in the first transition. Preliminary results indicate that the timing of age transitions can be manipulate with single-gene RNAi experiments. We are performing a detailed analysis by qPCR of the changes in gene expression of all of the the known small heat-shock protein genes in C. elegans associated with aging transitions. We are investigating the hypothesis that gene families may have members specific to certain age-states that get swapped out during these aging transitions. In current work, we are expanding the qPCR studies of small heat-shock family genes to other important gene families that undergo transcriptional regulation across these aging transitions.